Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.

Today the structure of photosystem II, which is the enzyme responsible for the evolution of molecular oxygen by plants, algae and cyanobacteria, is known up to a resolution of about 3.0 Å in cyanobacteria (Loll et al., 2005). Photosystem II of higher plants, which shows some differences compared to the photosystem II of cyanobacteria, is not resolved in such high detail, yet (8-10 Å) (Rhee et al., 1998; Hankamer et al., 2001a). Therefore, the molecular structure of PSII of higher plants and its adjacent antenna complexes remains in the focus of the current research. One of the major problems when working with photosystem II is its relative instability during isolation. Together with the antenna proteins and several other proteins, some of which still have an unclear function, PSII forms a huge multi-protein-complex, which tends to fall apart during classical preparation methods. In order to achieve a faster and milder method of purification for PSII, four different His-tags have been added to one of the subunits of PSII. The gene targeted in this study is called psbE and codes for the &#945;-chain of cytochrome b559, an integral part of PSII. The gene for PsbE is encoded in the chloroplast genome. The His-tags, which were employed in this work, consist of six or ten consecutive histidine aminoacid residues, which were fused to the N-terminus of the protein, either with or without a cleavage site for the protease “Factor Xa”. The N-terminus of PsbE is located on the more accessible stromal side of the thylakoid membrane. After inserting the psbE gene in a vector plasmid, in which the recognition site for the restriction endonuclease SacI had been eliminated, the different His-tags were generated by PCR with purposefully altered primers. In a final cloning step, a gene, which confers resistance to the antibiotics spectinomycin and streptomycin, was added to the DNA construct. Subsequently, the so-called biolistic transformation method (“gene gun”) was applied to introduce this genetically engineered plasmid DNA to Nicotiana tabacum chloroplasts (Bock & Hagemann, 2000). Through the processes of homologous recombination that take place in the chloroplast, the plastid encoded wildtype psbE gene was replaced by its His-tag containing counterparts. After several rounds of regenerating plants on antibiotic-containing medium, successful transformation was confirmed through PCR methods. By self fertilisation of fully regenerated plants, seeds were produced from tobacco strains, which carried only the mutated psbE gene. Plants cultivated from these seeds showed no distinctive phenotype under the chosen growth conditions, in respect to wildtype plants. The presence of the His-tag in this F1 generation was again confirmed with PCR methods. Measurements of oxygen evolution and pulse amplitude modulated fluorescence (PAM), carried out with preparations of wildtype and transgenic tobacco strains, revealed no differences for photochemical or non-photochemical quenching between both types. However, the oxygen evolution capacity of transgenic tobacco thylakoids compared to the wildtype was significantly reduced, although the chlorophyll content in relation to the leaf area was almost identical. This hints at a reduced amount of photosystem II complexes in the thylakoid membranes of transgenic tobacco. This alteration could be related to the mutation of cytochrome b559, because, amongst other functions, this subunit was shown to be important for the assembly of photosystem II (Morais et al., 1998). If solubilised thylakoid preparations of His-tagged plant strains were applied to a Ni-NTA column, photosystem II was selectively bound to the matrix. After washing away most of the contaminations, photosystem II core complexes could be eluted with imidazole-containing buffer. Photosystem II prepared in this way, displayed a drastic reduction of the peripheral light-harvesting complexes (LHCI & LHCII) and photo-system I reaction centres. This could be demonstrated by the loss of chlorophyll b and xanthophyll bands (LHCs) in absorption spectra, a small blue-shift of the chlorophyll a Qy absorption (PSI) and the respective band patterns in polyacrylamide gel electro-phoresis. The photosystem II complexes prepared in this way can now be put to use in different structural studies, like two-dimensional or three-dimensional crystallisation and spectroscopic measurements. Another photosynthetic pigment-protein complex of interest is the fucoxanthin-chlorophyll a/c-binding protein of diatoms, because eukaryotic algae, like diatoms, are important factors of oceanic ecosystems and account for a large part of marine biomass production. In order to facilitate ultra-fast time-resolved transient absorption spectroscopy and subsequent modelling of the kinetic traces, FCPs were prepared by sucrose-gradient ultra-centrifugation and their pigment stoichiometries determined by HPLC. Combining the spectroscopic data (Papagiannakis et al., 2005) with protein sequence alignments (Eppard & Rhiel, 1998) and the structure of the homologous higher plant LHCIIb (Kühlbrandt et al., 1994), a hypothetical model for the structure of FCP could be proposed (Fig. IV.3)